Tissue plasminogen activator molecules are found in a partially processed form following secretion from a eukaryotic host cell. The partially processed form results from the incomplete removal of the propeptide region from the amino-terminus of the mature t-PA molecule. During incubation and purification, proteolytic cleavage of the remainder of the propeptide region results in the accumulation of the fully processed form. Thus, a mixture of fully and partially processed t-PA molecules are present following purification of the t-PA molecules from the culture medium. According to the present invention, the heterogeneity at the amino-terminus is eliminated by providing a novel propeptide region that is capable of being uniformly cleaved by a cell associated protease. The expression vectors of the present invention provide a convenient means of producing the novel propeptide-containing forms of tissue plasminogen activator in eukaryotic host cells. Following expression, these novel t-PA molecules are uniformly cleaved by a cell associated protease and are secreted into the host cell culture medium in the fully processed form.
Plasminogen activators are a unique class of enzymes that convert plasminogen to its active enzymatic form, plasmin. Plasmin is a serine protease that degrades the fibrin networks of blood clots. Several plasminogen activators are currently being used as in vivo fibrinolytic agents in the treatment of acute myocardial infarction. One of these plasminogen activators, tissue plasminogen activator, has a significantly enhanced ability to activate plasminogen in the presence of fibrin. Thus, when used as an in vivo fibrinolytic agent, t-PA is more selective for fibrin clots.
Human t-PA is a multi-domain serine protease with five distinct structural domains that make up the 527 amino acids of the mature t-PA molecule. The molecule is synthesized as a one-chain polypeptide that can be converted to a two-chain form by a plasmin-mediated cleavage at Arginine 275-Isoleucine 276.
The amino-terminal portion of the molecule contains a disulfide-linked loop referred to as the finger domain. This domain is highly homologous to the finger domain of fibronectin which provides that molecule with fibrin-binding properties. The second domain, called the EGF domain, is highly homologous with epidermal growth factor (EGF). Similar growth factor domains occur in serine proteases such as urokinase, protein C and clotting factors IX and X. The third and fourth domains are highly disulfide-linked structures referred to as kringles (K1 and K2). Similar homologous kringle structures are present in plasma proteins such as plasminogen and prothrombin. There is conflicting evidence as to whether both kringle domains or just the second kringle domain are involved in the fibrin-mediated activation of plasminogen. The fifth domain, located at the carboxy-terminus, is the serine protease domain. The serine protease domain is homologous to similar domains in urokinase, plasma serine clotting proteases and trypsin.
The DNA encoding the amino-terminal end of tissue plasminogen activator encodes a precursor region of 35 amino acids (amino acids -1 to -35). The amino acid sequence (SEQ ID NO:1) of this precursor region is: ##STR1## wherein Arg is Arginine, Asn is Asparagine, Asp is Aspartic acid, Cys is Cysteine, Gln is Glutamine, Glu is Glutamic Acid, Gly is Glycine, His is Histidine, Ile is Isoleucine, LEU is Leucine, Lys is Lysine, Met is Methionine, Phe is Phenylalanine, Pro is Proline, Ser is Serine, Thr is Threonine, Trp is Tryptophan, Tyr is Tyrosine, and Val is Valine. The DNA and amino acid sequence of human tissue plasminogen activator was described by Pennica et al., 1983, Nature 301:214.
Amino acids -13 to -35 are hydrophobic amino acids comprising a signal sequence that directs secretion of the t-PA molecule from the host cell (Vehar et al., 1984, BIO/TECHNOLOGY 2(12):1051). This signal sequence is cleaved from the t-PA molecule during the secretion process, yielding a t-PA molecule with a 12 amino acid propeptide extension at the amino-terminal end. When t-PA is produced by melanoma cells using cell culture techniques the t-PA molecules isolated from the resulting culture medium exist in two forms (Wallen et al., 1983, Eur. J. Biochem. 132:681-686). One form is a t-PA molecule from which 9 of the 12 amino acids of the propeptide region have been cleaved resulting in a t-PA molecule with the tripeptide amino-terminal extension H.sub.2 N-Gly-Ala-Arg. A second form is a fully processed mature t-PA from which the entire propeptide region has been cleaved (Vehar et al., 1984).
Analysis of recombinantly produced t-PA variants from cultures incubated for short periods of time indicate that the t-PA molecules are secreted in the partially processed form (Burck et al., 1990, Journal of Biological Chemistry 265(9):5170). However, analysis of the t-PA molecules after longer periods of incubation or following purification of the molecules from the culture medium indicated that the remaining amino-terminal propeptide extension is cleaved from a portion of the molecules. Thus, the production of t-PA molecules is plagued by the presence of a heterogeneous final product consisting of both partially and fully processed forms.
It is the purpose of the present invention to provide a method for the recombinant production of t-PA or derivatives thereof by use of propeptide regions that are uniformly cleaved upon secretion from the cell. Use of this method provides a homogeneous population of t-PA molecules produced by recombinant DNA methods.
For purposes of the present invention, as disclosed and claimed herein, the following terms are defined below.
ApR--the ampicillin-resistant phenotype or gene conferring same.
Cloning--the process of incorporating a segment of DNA into a recombinant DNA cloning vector.
E1A--an immediate-early gene product of adenovirus which can activate a poly-GT element to express enhancer activity and can also activate the BK virus enhancer.
ep--a DNA segment comprising the SV40 early promoter of the T-antigen gene, the T-antigen binding sites, and the SV40 origin of replication.
Eukaryotic promoter--any DNA sequence that functions as a promoter in eukaryotic cells.
GBMT transcription control unit--a modified transcription control unit which comprises the P2 enhancer element of BK virus spaced closely to the upstream regulatory element of the major late promoter of adenovirus (MLTF), the adenovirus-2 major late promoter and a poly-GT element positioned to stimulate said promoter and a DNA sequence encoding the spliced tripartite leader of adenovirus-2. The GMBT transcription control unit is best exemplified by the approximately 900 base pair HindIII cassette found in plasmid pGTC which is found in E. coli K-12 AG1/pGTC (NRRL B-18593).
GT--enhancer system--any poly-GT element linked to a promoter, such as MLP, in which the poly-GT element does not itself possess enhancer activity but is activated as an enhancer by an immediate-early gene product of a large DNA virus, such as the E1A gene product or by any similarly activating viral gene product.
HmR--the hygromycin-resistant phenotype or gene conferring same.
IVS--DNA encoding an intron, also called an intervening sequence.
Large DNA virus--a virus that infects eukaryotic cells and has a genome greater than .about.10 kb in size, i.e., any of the pox viruses, adenoviruses, and herpes viruses.
MLP--the major late promoter of adenovirus, which is also referred to herein as the adenovirus late promoter, adenovirus-type-2 late promoter, or Ad2 late promoter.
MLTF binding site--the site in adenovirus DNA where the major late transcription factor (MLTF) binds; the MLTF is required for MLP activity.
NeoR--the neomycin resistance-conferring gene, which can also be used to confer G418 resistance in eukaryotic host cells.
ori--a plasmid origin of replication.
pA--a DNA sequence encoding a polyadenylation signal.
Poly-GT element--a DNA sequence of (GT).sub.n --(CA).sub.n, which is illustrated herein by a sequence where n is 21, but which can also refer to sequences of varying lengths where n is greater or less than 21, and may refer to chemically synthesized (GT).sub.n -(CA).sub.n sequences or human genomic DNA fragments containing a (GT).sub.n -(CA).sub.n tract.
Promoter--a DNA sequence that directs transcription of DNA into RNA.
Recombinant DNA Cloning Vector--any autonomously replicating or integrating agent that comprises a DNA molecule to which one or more additional DNA segments can be or have been added.
Recombinant DNA Expression Vector--any recombinant DNA cloning vector comprising a promoter and associated insertion site, into which a DNA sequence that encodes a useful product can be inserted and expressed.
Recombinant DNA Vector--any recombinant DNA cloning or expression vector.
Replicon--any DNA sequence that controls the replication of a recombinant DNA vector.